ES2629630T3 - Treatment of diseases related to erythropoietin (EPO) by inhibiting the natural antisense transcript to EPO - Google Patents

Abstract

An oligonucleotide that targets a natural erythropoietin antisense transcript (EPO) for use as a therapeutic compound, where the oligonucleotide modulates expression of the erythropoietin (EPO) gene and where the natural antisense transcript has the nucleic acid sequence as set forth in SEQ ID NO: 2.

Description

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As used herein, the term "target nucleic acid" encompasses DNA, RNA (comprising pre-mRNA and RNA) transcribed from said DNA, and also cDNA derived from said RNA, coding, non-coding sequences, sense or antisense polynucleotides. Specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of the function of a target nucleic acid by compounds, which hybridize specifically with it, is generally referred to as "antisense." The DNA functions with which they will interfere include, for example, replication and transcription. The RNA functions with which they will interfere include all vital functions such as, for example, RNA translocation to the protein translation site, RNA protein translation, RNA splicing to give one or more mRNA species, and catalytic activity which can be coupled in or facilitated by RNA. The overall effect of such interference with the functions of the target nucleic acid is the modulation of the expression of a coded product or oligonucleotides.

"IRNA" RNA interference is mediated by double stranded RNA molecules (dsRNA) that have sequence-specific homology with their "target" nucleic acid sequences (Caplen, NJ, et al. (2001) Proc. Natl. Acad. Sci USA 98: 9742-9747). In certain cases of the present disclosure, the mediators are duplexes of "small interfering" RNA (siRNA) of 5-25 nucleotides. The siRNAs are derived from the processing of dsRNA by an RNAse enzyme known as Dicer (Bernstein, E., et al. (2001) Nature 409: 363-366). SiRNA duplex products are used in a multiprotein siRNA complex called RISC (RNA-Induced Silencer Complex). Without wishing to adhere to any particular theory, it is then believed that an RISC is guided to a target nucleic acid (suitably mRNA), in which the siRNA duplex interacts in a specific sequence way to mediate cleavage in a catalytic way ( Bernstein, E., et al. (2001) Nature 409: 363-366; Boutla, A., et al. (2001) Curr. Biol. 11: 1776-1780). Small interfering RNAs that can be used in accordance with the present disclosure can be synthesized and used according to procedures that are well known in the art and that will be familiar to the person skilled in the art. Small interfering RNAs for use in the methods of the present disclosure suitably comprise between about 1 and about 50 nucleotides (nt). In the examples of non-limiting aspects, the siRNAs may comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

The selection of appropriate oligonucleotides is facilitated using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that show an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of astringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of oligonucleotides that show a high degree of complementarity with target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity with corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable freedom in the selection of appropriate regions of genes for use in the present disclosure.

By "enzymatic RNA" is indicated an RNA molecule with enzymatic activity (Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymatic nucleic acids (ribozymes) act by first binding to a target RNA. Said binding occurs through the target binding part of an enzymatic nucleic acid that is maintained in close proximity to an enzymatic part of the molecule that acts to cleave the target RNA. In this way, the enzyme nucleic acid first recognizes and then binds to target RNA by base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

By "decoy RNA" is indicated an RNA molecule that mimics the natural binding domain for a ligand. The decoy RNA competes, therefore, with the natural binding target for the binding of a specific ligand. For example, it has been shown that overexpression of HIV transactivation response (TAR) RNA can act as a "decoy" and efficiently binds to HIV tat protein, thereby preventing it from binding to TAR sequences. encoded by HIV RNA (Sullenger et al. (1990) Cell, 63, 601-608). This is indicated to be a specific example. Those skilled in the art will recognize that this is just an example, and other aspects can easily be generated using techniques generally known in the art.

As used herein, the term "monomers" normally indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides that vary in size between a few.

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others. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene among individuals of a given species. Polymorphic variants can also encompass "single nucleotide polymorphisms" (SNPs), or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNP may be indicative of, for example, a certain population with a propensity for a pathology, that is, susceptibility to resistance.

Derived polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl or amino group. The derivatives, for example, derived oligonucleotides, may comprise non-naturally occurring parts, such as altered sugar moieties or inter-sugar bonds. By way of example, these include phosphorothioate and other sulfur-containing species that are known in the art. The derived nucleic acids may also contain tags, which include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles and the like.

A "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulphation, reduction / alkylation, acylation, chemical coupling or gentle formalin treatment. A derivative can also be modified to contain a detectable label, both directly and indirectly, which includes, but is not limited to, a radioisotope, fluorescent and enzymatic label.

As used herein, the term "animal" or "patient" is intended to include, for example, humans, sheep, elk, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats , dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.

"Mammal" covers warm-blooded mammals that are normally under medical attention (for example, humans and domesticated animals). Examples include felines, canines, equines, bovines and humans, as well as only humans.

"Treat" or "treatment" covers the treatment of a pathology in a mammal, and includes: (a) preventing the occurrence of pathology in a mammal, in particular, when said mammal is predisposed to the pathology, but has not yet been diagnosed to have it; (b) inhibit pathology, for example, stop development; and / or (c) alleviate the pathology, for example, causing the regression of the pathology until a desired assessment criterion is reached. Treating also includes the improvement of a symptom of a disease (for example, reducing pain or discomfort), where such improvement may or may not directly affect the disease (for example, cause, transmission, expression, etc.).

Compositions and molecules of polynucleotides and oligonucleotides

Targets: In one aspect, the targets comprise erythropoietin (EPO) nucleic acid sequences, including without limitation non-coding and / or coding, sense and / or antisense sequences associated with EPO.

Erythropoietin (EPO) is a member of the hematopoietic growth factor family that acts as a hormone. EPO regulates the production of erythrocytes (erythropoiesis) and keeps the body mass of erythrocytes at an optimal level. EPO production is stimulated by a reduced oxygen content in the renal arterial circulation, mediated by a transcription factor that is sensitive to oxygen. EPO is mainly produced by cells of the peritubular capillary endothelium of the kidney. Secreted EPO binds to EPO receptors on the surface of bone marrow-erythroid precursors and results in rapid replication and maturation for functional erythrocytes. This stimulation results in a rapid increase in erythrocyte counts and a consequent increase in hematocrit (% of red blood cells) (D'Andrea et al. (1989) Cell 57: 277-285; Lodish et al. (1995) Cold Spring Harb Symp Quant Biol 60: 93-104).

Erythropoietin stimulates the bone marrow to produce more erythrocytes. The resulting increase in erythrocytes increases the ability to transport oxygen in the blood. As the main regulator of erythrocyte production, the main functions of erythropoietin are: to promote the development of erythrocytes; Start the synthesis of hemoglobin, the molecule inside the erythrocytes that carries oxygen.

EPO stimulates the mitotic division and differentiation of erythrocyte precursor cells and therefore ensures the production of erythrocytes. It occurs in the kidneys when hypoxic conditions prevail. During the differentiation of erythrocyte precursor cells induced by EPO, there is an induction of globulin synthesis and an increase in heme complex synthesis and in the number of ferritin receptors. This makes it possible for the cell to take more ions and synthesize functional hemoglobin. Hemoglobin of mature red blood cells

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U.S. Pat. with no. 5,688,679 also reported the use of supernatants from selected cell lines for competitive radioimmunoassay assays using a polyvalent human antieritropoietin rabbit antiserum (J.Cell.Physiol. 118: 87-96, 1984). Erythropoietin can also be detected using other immunological procedures, for example: Western blotting and immunofluorescence.

In addition, the same patent describes how to perform an assay of erythropoietin secreted in cell supernatant for proliferative effects and other bone marrow progenitor cells. Erythropoietin can be analyzed for its effect on a variety of progenitors from mouse and human marrow that include erythroid colony forming cells (CFU-E), erythroid discharge forming cells (BFU-E), granulocyte precursors. macrophage (CFU-GM), and mixed cell colony forming cells (CFU-Mixed) (J.Cell.Physiol.Suppl. 1: 79-85, 1982; J.Cell.Physiol. 118: 87-96, 1984 ).

It has also been reported that erythropoietin alters other cell types in addition to those of the erythrocytic line. For example, Lifshitz, et al., 2009, "Non-erythroid activities of erythropoietin: Functional effects on murine dendritic cells," Mol Immunol. 46 (4): 713-21, also reports experiments in vivo in mice that have been injected with EPO and in transgenic mice that overexpress human EPO that show an enlarged DC population in spleen with a cell surface expression greater than CD80 and CD86. Prutchi, et al., 2008, "erythropoietin effects on dendritic cells: potential mediators in its function as an immunomodulator?" Hematol Exp. 36 (12): 1682-90, indicated that when applied in vitro, EPO increases the percentage of peripheral blood DC and monocyte-derived DC (MoDC) that express CD80 and CD86 costimulatory molecules.

In a preferred aspect, the oligonucleotides are specific for EPO polynucleotides, which includes, without limitation, non-coding regions. EPO targets comprise variants of EPO; EPO mutants, including SNP; EPO non-coding sequences; alleles, fragments and the like. Preferably, the oligonucleotide is an antisense RNA molecule.

According to aspects of the disclosure, the target nucleic acid molecule is not limited to EPO polynucleotides alone, but extends to any of the isoforms, receptors, homologs, non-coding regions and the like of EPO.

In another preferred aspect, an oligonucleotide is directed to a natural antisense sequence (natural antisense to the coding and non-coding regions) of EPO targets, including, without limitation, variants, alleles, homologues, mutants, derivatives, fragments and sequences complementary to these. Preferably the oligonucleotide is an antisense RNA or DNA molecule.

In another preferred aspect, the oligomeric compounds of the present disclosure also include variants in which a different base is present in one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenine, variants containing thymidine, guanosine, cytidine or other natural or unnatural nucleotides may be produced in this position. This can be done in any of the positions of the antisense compound. These compounds are then tested using the procedures described herein to determine their ability to inhibit the expression of a target nucleic acid.

In some aspects, the homology, sequence identity or complementarity, between the antisense compound and the target, is from about 50% to about 60%. In some aspects, the homology, sequence identity or complementarity is from about 60% to about 70%. In some aspects, the homology, sequence identity or complementarity is from about 70% to about 80%. In some aspects, the homology, sequence identity or complementarity is from about 80% to about 90%. In some aspects, homology, sequence identity

or complementarity is approximately 90%, approximately 92%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99% or approximately 100%.

An antisense compound is specifically hybridizable when the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to produce a loss of activity, and there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to acid sequences. non-target nucleic under conditions where specific binding is desired. Such conditions include, that is, physiological conditions in the case of in vivo tests or therapeutic treatment, and conditions under which the tests are performed in the case of in vitro tests.

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nucleotides between the translation termination codon and the 3 'end of an mRNA (or corresponding nucleotides in the gene). The 5 'cap site of an mRNA comprises an N7-methylated guanosine residue attached to the plus 5' residue of the mRNA via a 5'-5 'triphosphate bond. The 5 'cap region of an mRNA is considered to include the 5' cap structure itself, in addition to the first 50 nucleotides adjacent to the cap site. Another target region for this disclosure is the 5 'hood region.

Although some eukaryotic mRNA transcripts are translated directly, many contain one or more regions, known as "introns," which are cleaved from a transcript before it is translated. The remaining regions (and therefore translated) are known as "exons" and are spliced together to form a continuous mRNA sequence. In one aspect, the choice of target splicing sites, that is, intron-exon splices or exon-intron splices, is particularly useful in situations where aberrant splicing participates in the disease, or in which an excess production of a particular splicing product participates in the disease. An aberrant fusion splice due to transposition or deletion is another aspect of a target site. Transcribed mRNAs produced by the splicing process of two (or more) mRNAs from different gene sources are known as "fusion transcripts." Introns can be effectively chosen as targets using antisense compounds targeting, for example, DNA or pre-mRNA.

In another preferred aspect, the antisense oligonucleotides bind to coding and / or non-coding regions of a target polynucleotide and modulate the expression and / or function of the target molecule.

In another preferred aspect, the antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and / or function of the target molecule.

In another preferred aspect, the antisense oligonucleotides bind sense polynucleotides and modulate the expression and / or function of the target molecule.

Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants." More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA at their start or stop position and contain both intronic and exonic sequences.

Upon excision of one or more regions of exon or intron, or parts thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Therefore, mRNA variants are pre-mRNA variants processed and each single pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splicing variants". If splicing of the pre-mRNA variant does not occur, then the pre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals for start or stop transcription. Pre-mRNA and mRNA may have more than one initiation codon or termination codon. Variants that originate from a pre-mRNA or mRNA that use alternative initiation codons are known as "alternative initiation variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative termination codon are known as "alternative stop variants" of that pre-mRNA or mRNA. A specific type of alternative stop variant is the "polyA variant", in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts. which end in unique polyA sites. Within the context of the disclosure, the types of variants described herein are also aspects of target nucleic acids.

The locations in the target nucleic acid with which the antisense compounds hybridize are defined as at least a part of 5 nucleotides in length of a target region to which an active antisense compound is directed.

Although the specific sequences of certain target segments by way of example are set forth herein, one skilled in the art will recognize that they serve to illustrate and describe particular aspects within the scope of the present disclosure. Additional target segments are easily identifiable by a person skilled in the art in view of this disclosure.

It is considered that target segments of 5-100 nucleotides in length comprising a stretch of at least five

(5) consecutive nucleotides selected from within the illustrative preferred target segments are also suitable for addressing.

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Inactivation of its antisense counterpart could possibly mimic the action of a receptor agonist or a stimulating enzyme.

Strategy 2: In the case of concordant regulation, both antisense and sense transcripts could be concomitantly inactivated and thus achieve a synergistic reduction of conventional gene expression (sense). If, for example, an antisense oligonucleotide is used to achieve inactivation, then this strategy can be used to apply an antisense oligonucleotide directed to the sense transcript and another antisense oligonucleotide to the corresponding antisense transcript, or a single energy-symmetrical antisense oligonucleotide that simultaneously targets transcribed sense and antisense overlapping.

In accordance with the present disclosure, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference compounds (iRNAs) such as siRNA compounds, and others. oligomeric compounds that hybridize with at least a portion of the target nucleic acid and modulate its function. Therefore, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or they may be mimetics of one or more of these. These compounds may be single-stranded, double-stranded, circular or fork oligomeric compounds and may contain structural elements such as internal or terminal protrusions, mismatches or loops. Antisense compounds are routinely prepared linearly, but can be joined or otherwise prepared to be circular and / or branched. The antisense compounds may comprise constructs such as, for example, two hybridized chains to form a complete or partially double-stranded compound or a single chain with sufficient self-complementarity to allow hybridization and formation of a complete or partially double-stranded compound. The two chains can be linked internally, leaving the 3 'or 5' ends free or they can be linked to form a continuous fork or loop structure. The hairpin structure may contain a protuberant nucleotide at any of the 5 'or 3' ends that produces an extension of the single stranded character. The double stranded compounds may optionally comprise protruding nucleotides at the ends. Additional modifications may comprise conjugated groups attached to one of the ends, selected nucleotide positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two chains can be linked by a non-nucleic acid moiety or linker group. When formed from just one chain, the dsRNA can take the form of a self-complementary hairpin-like molecule that bends over itself to form a duplex. In this way, the dsRNA can be completely or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some cases, gene expression or function is positively regulated. When formed from two chains, or a single chain that takes the form of a self-complementary hairpin-like molecule that bends over itself to form a duplex, the two chains (or duplex-forming regions of a single chain ) are complementary RNA strands that mate with bases in the Watson-Crick mode.

Once introduced into a system, the compounds of the disclosure may cause the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid or may work by occupation-based mechanisms. In general, nucleic acids (including oligonucleotides) can be described as "DNA-like" (that is, they generally have one or more 2'-deoxy sugars and, generally, T bases instead of U) or "RNA-like" (is that is, they generally have one or more 2'-hydroxy sugars or sugars modified in 2 'and generally U bases instead of T). Nucleic acid helices can adopt more than one type of structure, most commonly forms A and B. It is believed that, in general, oligonucleotides that have a structure similar to form B are "DNA-like" and those that have structure similar to form A are "similar to RNA". In some (chimeric) cases, an antisense compound may contain both A-shaped and B-shaped regions. In another preferred aspect, the desired oligonucleotides or antisense compounds comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides that they comprise modified bonds, interference RNA (iRNA), small interfering RNA (siRNA); an interfering microRNA (miRNA); a small temporary RNA (tRNA); or a short hairpin RNA (hRNA); small RNA-induced gene activation (aRNA); Small activating RNA (siRNA), or combinations thereof.

DsRNAs can also activate gene expression, a mechanism that has been called "small RNA-induced gene activation" or aRNA. Gene promoters that target dsRNA induce the potent transcriptional activation of associated genes. The aRNA was demonstrated in human cells using synthetic dsRNAs, called "small activating RNAs" (siRNA). It is not currently known if the aRNA is conserved in other organisms.

It has been found that small double stranded RNA (dsRNA), such as small interfering RNA (siRNA) and microRNA (miRNA), is the trigger for an evolutionarily conserved mechanism known as

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Claims (1)

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